An Orthogonal Framework for Higher-Order Structural and In Vitro Functional Comparability of Chemically Synthesized Liraglutide Relative to an rDNA-Origin Reference Product.

PURPOSE: Synthetic peptide test products referencing recombinant-origin products present unique regulatory challenges in demonstrating higher-order structural (HOS) comparability beyond primary sequence sameness. Liraglutide, a lipidated glucagon-like peptide-1 (GLP-1) receptor agonist with defined self-association behavior, represents an important model for such assessments. METHODS: A head-to-head comparability study was performed between a chemically synthesized liraglutide product and its recombinant reference listed drug (RLD). An orthogonal analytical framework integrating spectroscopic, biophysical, nuclear magnetic resonance (NMR)-based, and in vitro functional methods was applied to assess secondary structure, supramolecular assembly, aggregation propensity, molecular conformational fingerprints, and biological activity. RESULTS: The test products and RLD demonstrated highly comparable structural and functional profiles across all evaluated dimensions. Secondary structural characteristics, NMR fingerprints, and spatial conformational features were highly consistent. Solution-state analyses showed comparable diffusion behavior and a dominant low-oligomeric assembly, with apparent aggregation numbers close to the hexamer-to-heptamer range and without evidence of abnormal aggregation or fibrillation. Functional studies further confirmed comparable GLP-1 receptor activity and stability profiles. CONCLUSIONS: Orthogonal structural and functional characterization can substantially reduce residual uncertainty for synthetic peptide generics referencing recombinant products and provides a practical framework for comparability assessment of complex peptide drugs.